This project has the objective of understanding the chemistry and structure of nucleic acids and relating this knowledge to the biological functions of these molecules. Methods used include chemical synthesis of defined sequence DNA fragments and of enzyme substrates, enzymatic synthesis of polynucleotides, study of nucleic acids by circular dichroism, ultraviolet, infrared, and nuclear magnetic resonance spectroscopy, study of thermal transitions and dependence of physical properties on solution conditions. Subjects of investigation include factors which determine the stability of helical complexes, specificity of nucleic acid interactions, location and affinity of binding sites. We have continued our collaborations on 2D NMR of DNA fragments containing restriction endonuclease recognition sequences. Resonance assignments have been obtained for all the nonexchangeable protons in the dodecanucleotide d(GAATTCGAATTC) and approximate sugar conformations and glycosidic dihedral angles determined. The molecule has a B-DNA conformation with both strands identical. A new heteronuclear 1H-31p shift correlation method was used to assign all of the 31P resonances in the oligonucleotide d(CATGCATm5CCATG). Information on nucleic acid mispairing is relevant to such important biological functions as mutation, gene expression and control, splicing, and feedback control. We have begun examination of a series of oligonucleotides small enough to permit analysis of structural and energetic changes caused by introduction of selected mispairings. An initial finding of importance is that the position of AG mispairing is crucial for its effect on helix stability. Surprisingly, two AG's replacing two CG's in the center of a dodecamer have little effect on Tm, whereas the same substitutions two positions removed from the center have a large effect. A similar result is observed with AI pairing. Preliminary work with AC mispairing shows that it is strongly destabilizing.